The mitochondrial theory of aging states that the accumulation of oxidative damage with age results in mitochondrial dysfunction, leading to altered energetics and initiation of cell death cascades. However, support for this chain of events is equivocal, despite several decades of intense research effort. Due to the lack of necessary tools to measure mitochondrial function in vivo, current approaches have typically focused on in vitro measurements of mitochondrial function, particularly the electron transport chain (ETC), making it necessary to extrapolate to the physiological state. To overcome this limitation, we have developed novel methods to directly measure mitochondrial function in vivo. We propose that reduced coupling of ATP synthesis to O2 consumption (P/O) is an important mechanism of mitochondrial dysfunction in aging muscle. We test this hypothesis in aim 1 by determining in vivo mitochondrial P/O, maximal ETC flux, capacity for ATP synthesis, and in vitro ETC activity in relation to the accumulation of oxidative damage in mouse skeletal muscle at four ages. In aim 2 we test the mechanistic link between oxidative damage and mitochondrial dysfunction in vivo using a transgenic mouse model that overexpresses an antioxidant enzyme in mitochondria to increase the resistance of mitochodnria to oxidative stress with age. Aim 3 tests the reversibility of the loss of mitochondrial function in aging muscle using exercise training to increase mitochondrial proliferation and turnover, thereby replacing damaged mitochondria. This proposal uses state of the art in vivo spectroscopy to address the controversy surrounding experimental evidence for the mitochondrial theory of aging. This mentored research plan will facilitate Dr. Marcinek's development as a gerontologic researcher and junior faculty member in the Department of Radiology at the University of Washington. The mentors for this proposal represent several decades of experience in their respective fields - George Martin and Peter Rabinovitch for the biology of aging and Kevin Conley and Martin Kushmerick for quantitative bioenergetics and in vivo spectroscopy. Internationally recognized programs in muscle metabolism and the biology of aging at the University of Washington make this environment ideally suited to a research career integrating the study of aging and metabolism.